Isoelectric focusing (IEF) is an electrophoretic technique that is commonly used for the analysis, separation and purification of various biological materials, and particularly proteins. Since many of the complex molecules of biological interest are amphoteric in nature, they are typically amenable to IEF separation. Gel electrophoresis is a process that is commonly used for protein and DNA analysis.
The separation of macromolecules, and particularly proteins, often is carried out by two-dimensional electrophoresis separation. The two-dimensional electrophoresis separation typically involves the sequential separation by isoelectric focusing of a sample in a gel tube followed by slab gel electrophoresis. The isoelectric focusing process in the gel tube is often referred to as first dimension separation.
In the first dimension separation, an isoelectric focusing gel, such as acrylamide, is placed or polymerized in a tube. The open ends of the tube are positioned in a tank with a buffer solution at each end of the tube. One end of the tube is positioned in a bath of a buffer solution such as sodium hydroxide solution. The other end of the tube is positioned in a bath of a second buffer solution such as a phosphoric acid solution. An electric current is applied to the two buffer solutions. The current together with ampholytes incorporated into the gel composition or titratable gel monomers incorporated into the gel, provides a pH gradient through the gel along the length of the tube. The sample to be analyzed is applied to a one end of the gel in the tube and an electric current is applied to an electrode in each of the buffer solutions. The molecules in the sample migrate through the gel under the influence of the electric potential until they reach their respective isoelectric point.
Slab gel electrophoresis, often referred to as second dimension separation, utilizes an electrophoresis gel molded between two glass plates. A gel strip or cylinder in which the protein sample has been resolved by the first dimension isoelectric focusing is placed along one edge of the slab gel. The ends of the gel slab are positioned in a buffer solution and an electric current is applied to each end of the gel. The proteins are then allowed to migrate through the gel slab under an applied voltage.
Charged detergents, such as sodium dodecyl sulfate, contained in the slab gel bind to the protein molecules. The detergents tend to unfold the protein molecules into rods having a length proportional to the length of the polypeptide chain and thus proportional to the molecular weight of the polypeptide. A protein complexed with a charged detergent is highly charged, which causes the protein-detergent complex to move in an applied electric field. When the slab gel, such as a polyacrylamide gel, functions as a sieve, the movement of the longer and higher molecular weight molecules is retarded compared to the shorter, lower molecular weight molecules.
Electrophoresis separation is generally labor intensive since numerous samples are run simultaneously. Generally, the gel tubes are prepared and placed in a suitable tank of buffer solutions. The protein samples are then manually placed on the end of a gel tube. When hundreds of protein samples are prepared daily for isoelectric focusing, the manual steps significantly increase the time requirements for performing the first dimension separation.
The resolution of the separation methods are sufficient to separate at least 150 proteins from a mixture. The first dimension isoelectric focusing separation followed by the second dimension SDS electrophoresis separation can result in the resolution of as many as 22,000 proteins from a single sample. A critical step in obtaining high resolution two-dimensional electrophoresis is to coordinate the first dimension separation with the second dimension separation.
The gel slab is removed from the glass plates and immersed in a series of baths containing various staining agents. Typically, the gel slabs are manually transferred from a stain bath to various fixing solutions and rinsing solutions. After the second dimension electrophoresis separation, the gel is developed to stain the proteins which appear as a spot on the gel. Thereafter, a gel spot can be identified, removed from the slab, and analyzed.
Various automated devices are known for performing various analysis processes of proteins and DNA. One example is disclosed in U.S. Pat. No. 5,865,975 to Bishop. The disclosed system uses an automated protein and DNA gene fragments analyzing machine where electrophoresis cells are robotically inserted into an electrophoresis housing for producing electrophoretic migration of the protein in one dimension. The robotic assembly rotates the cells 90° to enable separation of the fragments vertically in a second dimension.
The gel slabs are made of a flexible gel and care must be taken to prevent damaging or tearing the gel. During handling and manipulating, the gel slab adheres to surfaces that it contacts. As the gel is pulled from the surface, the gel can tear or stretch. Various devices have been proposed for handling and manipulating gel slabs. However, these devices have experienced only limited success. Accordingly, there is a continuing need in the industry for improved methods and devices for handling and processing electrophoresis gels.